The tonoplast localized protein PtNPF1 participates in the regulation of nitrogen response in diatoms.

Diatoms are a highly successful group of phytoplankton, well adapted also to oligotrophic environments and capable of handling nutrient fluctuations in the ocean, particularly nitrate. The presence of a large vacuole is an important trait contributing to their adaptive features. It confers diatoms the ability to accumulate and store nutrients, such as nitrate, when they are abundant outside and then to reallocate them into the cytosol to meet deficiencies, in a process called luxury uptake. The molecular mechanisms that regulate these nitrate fluxes are still not known in diatoms. In this work, we provide new insights into the function of Phaeodactylum tricornutum NPF1, a putative low-affinity nitrate transporter. To accomplish this, we generated overexpressing strains and CRISPR/Cas9 loss-of-function mutants. Microscopy observations confirmed predictions that PtNPF1 is localized on the vacuole membrane. Furthermore, functional characterizations performed on knock-out mutants revealed a transient growth delay phenotype linked to altered nitrate uptake. Together, these results allowed us to hypothesize that PtNPF1 is presumably involved in modulating intracellular nitrogen fluxes, managing intracellular nutrient availability. This ability might allow diatoms to fine-tune the assimilation, storage and reallocation of nitrate, conferring them a strong advantage in oligotrophic environments.

Transcriptional chronology reveals conserved genes involved in pennate diatom sexual reproduction.

Sexual reproduction is a major driver of adaptation and speciation in eukaryotes. In diatoms, siliceous microalgae with a unique cell size reduction-restitution life cycle and among the world's most prolific primary producers, sex also acts as the main mechanism for cell size restoration through the formation of an expanding auxospore. However, the molecular regulators of the different stages of sexual reproduction and size restoration are poorly explored. Here, we combined RNA sequencing with the assembly of a 55 Mbp reference genome for Cylindrotheca closterium to identify patterns of gene expression during different stages of sexual reproduction. These were compared with a corresponding transcriptomic time series of Seminavis robusta to assess the degree of expression conservation. Integrative orthology analysis revealed 138 one-to-one orthologues that are upregulated during sex in both species, among which 56 genes consistently upregulated during cell pairing and gametogenesis, and 11 genes induced when auxospores are present. Several early, sex-specific transcription factors and B-type cyclins were also upregulated during sex in other pennate and centric diatoms, pointing towards a conserved core regulatory machinery for meiosis and gametogenesis across diatoms. Furthermore, we find molecular evidence that the pheromone-induced cell cycle arrest is short-lived in benthic diatoms, which may be linked to their active mode of mate finding through gliding. Finally, we exploit the temporal resolution of our comparative analysis to report the first marker genes for auxospore identity called AAE1-3 ("Auxospore-Associated Expression"). Altogether, we introduce a multi-species model of the transcriptional dynamics during size restoration in diatoms and highlight conserved gene expression dynamics during different stages of sexual reproduction.

Gene expression during the formation of resting spores induced by nitrogen starvation in the marine diatom Chaetoceros socialis.

BACKGROUND: Dormancy is widespread in both multicellular and unicellular organisms. Among diatoms, unicellular microalgae at the base of all aquatic food webs, several species produce dormant cells (spores or resting cells) that can withstand long periods of adverse environmental conditions. RESULTS: We present the first gene expression study during the process of spore formation induced by nitrogen depletion in the marine planktonic diatom Chaetoceros socialis. In this condition, genes related to photosynthesis and nitrate assimilation, including high-affinity nitrate transporters (NTRs), were downregulated. While the former result is a common reaction among diatoms under nitrogen stress, the latter seems to be exclusive of the spore-former C. socialis. The upregulation of catabolic pathways, such as tricarboxylic acid cycle, glyoxylate cycle and fatty acid beta-oxidation, suggests that this diatom could use lipids as a source of energy during the process of spore formation. Furthermore, the upregulation of a lipoxygenase and several aldehyde dehydrogenases (ALDHs) advocates the presence of oxylipin-mediated signaling, while the upregulation of genes involved in dormancy-related pathways conserved in other organisms (e.g. serine/threonine-protein kinases TOR and its inhibitor GATOR) provides interesting avenues for future explorations. CONCLUSIONS: Our results demonstrate that the transition from an active growth phase to a resting one is characterized by marked metabolic changes and provides evidence for the presence of signaling pathways related to intercellular communication.

The pennate diatom Pseudo-nitzschia multistriata as a model for diatom life cycles, from the laboratory to the sea.

Phytoplankton dynamics are regulated by external cues, such as light and nutrients, as well as by biotic interactions and endogenous controls linked to life cycle characteristics. The planktonic pennate diatom Pseudo-nitzschia multistriata, with a heterothallic mating system with two opposite mating types (MTs), represents a model for the study of diatom life cycles. P. multistriata is a toxic species, able to produce the neurotoxin domoic acid. First described in Japan in 1993, it was detected at the long-term monitoring station MareChiara (Gulf of Naples, Italy) in 1995. Since then, P. multistriata has been reported from several worldwide coastal sites. A large body of knowledge has been produced on its ecology, genetic diversity, and life cycle characteristics. The availability of these data, the ecological relevance of the Pseudo-nitzschia genus, and its controllable life cycle with a short generation time made it an ideal species to develop a genetic model system for diatoms. To enable functional studies, a 59 Mb genome sequence and several transcriptomic data were produced, and genetic transformation was optimized. These tools allowed the discovery of the first mating-type determining gene for diatoms. Gene expression studies and metabolomics analyses defined genes and molecules underpinning different phases of the process of sexual reproduction. This model system, developed to explore the genetics of diatom life cycles, offers the opportunity to parallel experimental observations in the laboratory using in situ meta-omics analyses along space and time, empowering knowledge on the biology and ecology of the genus.

Silencing of a Pseudo-nitzschia arenysensis lipoxygenase transcript leads to reduced oxylipin production and impaired growth.

Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists. Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo- and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15. PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms. The presence in the genome of a single LOX gene, whose downregulation impairs both 12- and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms.

Highly Valuable Polyunsaturated Fatty Acids from Microalgae: Strategies to Improve Their Yields and Their Potential Exploitation in Aquaculture.

Microalgae have a great potential for the production of healthy food and feed supplements. Their ability to convert carbon into high-value compounds and to be cultured in large scale without interfering with crop cultivation makes these photosynthetic microorganisms promising for the sustainable production of lipids. In particular, microalgae represent an alternative source of polyunsaturated fatty acids (PUFAs), whose consumption is related to various health benefits for humans and animals. In recent years, several strategies to improve PUFAs' production in microalgae have been investigated. Such strategies include selecting the best performing species and strains and the optimization of culturing conditions, with special emphasis on the different cultivation systems and the effect of different abiotic factors on PUFAs' accumulation in microalgae. Moreover, developments and results obtained through the most modern genetic and metabolic engineering techniques are described, focusing on the strategies that lead to an increased lipid production or an altered PUFAs' profile. Additionally, we provide an overview of biotechnological applications of PUFAs derived from microalgae as safe and sustainable organisms, such as aquafeed and food ingredients, and of the main techniques (and their related issues) for PUFAs' extraction and purification from microalgal biomass.

A Metabolomics Exploration of the Sexual Phase in the Marine Diatom Pseudo-nitzschia multistriata.

Pseudo-nitzschia multistriata is a planktonic marine diatom with a diplontic life cycle comprising a short sexual phase, during which gametes are produced following the encounter of two diploid cells of opposite mating type (MT). Gene expression studies have highlighted the presence of substantial changes occurring at the onset of sexual reproduction. Herein, we have hypothesized that the amount and nature of cellular metabolites varies along the mating process. To capture the metabolome of Pseudo-nitzschia multistriata at different harvesting times in an unbiased manner, we undertook an untargeted metabolomics approach based on liquid chromatography-tandem mass spectrometry. Using three different extraction steps, the method revealed pronounced differences in the metabolic profiles between control cells in the vegetative phase (MT+ and MT-) and mixed strains of opposite MTs (cross) undergoing sexual reproduction. Of the 2408 high-quality features obtained, 70 known metabolites could be identified based on in-house libraries and online databases; additional 46 features could be classified by molecular networking of tandem mass spectra. The reduction of phytol detected in the cross can be linked to the general downregulation of photosynthesis during sexual reproduction observed elsewhere. Moreover, the role of highly regulated compounds such as 7-dehydrodesmosterol, whose changes in abundance were the highest in the experiment, oleamide, ectoine, or trigonelline is discussed.

Correction to: Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta.

Following the publication of this article [1], the authors reported that the link to Additional file 11 linked to the wrong set of data. The correct supplementary data is provided in this Correction article (Additional file 11).

Marine Natural Products from Microalgae: An -Omics Overview.

Over the last decade, genome sequences and other -omics datasets have been produced for a wide range of microalgae, and several others are on the way. Marine microalgae possess distinct and unique metabolic pathways, and can potentially produce specific secondary metabolites with biological activity (e.g., antipredator, allelopathic, antiproliferative, cytotoxic, anticancer, photoprotective, as well as anti-infective and antifouling activities). Because microalgae are very diverse, and adapted to a broad variety of environmental conditions, the chances to find novel and unexplored bioactive metabolites with properties of interest for biotechnological and biomedical applications are high. This review presents a comprehensive overview of the current efforts and of the available solutions to produce, explore and exploit -omics datasets, with the aim of identifying species and strains with the highest potential for the identification of novel marine natural products. In addition, funding efforts for the implementation of marine microalgal -omics resources and future perspectives are presented as well.

Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom.

Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo-nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex-related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein-coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis-related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient-replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction.

Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta.

BACKGROUND: Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. RESULTS: The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. CONCLUSIONS: Our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.

Oral-facial-digital type I protein is required for primary cilia formation and left-right axis specification.

The oral-facial-digital type I (OFD1) syndrome (OMIM 311200) is a human developmental disorder; affected individuals have craniofacial and digital abnormalities and, in 15% of cases, polycystic kidney. The disease is inherited as an X-linked dominant male-lethal trait. Using a Cre-loxP system, we generated knockout animals lacking Ofd1 and reproduced the main features of the disease, albeit with increased severity, possibly owing to differences of X inactivation patterns between human and mouse. We found failure of left-right axis specification in mutant male embryos, and ultrastructural analysis showed a lack of cilia in the embryonic node. Formation of cilia was defective in cystic kidneys from heterozygous females, implicating ciliogenesis as a mechanism underlying cyst development. In addition, we found impaired patterning of the neural tube and altered expression of the 5' Hoxa and Hoxd genes in the limb buds of mice lacking Ofd1, suggesting that Ofd1 could have a role beyond primary cilium organization and assembly.

The dynamics of sexual phase in the marine diatom Pseudo-nitzschia multistriata (Bacillariophyceae).

Sexual reproduction represents a fundamental phase in the life cycle of diatoms, linked to both the production of genotypic diversity and the formation of large-sized initial cells. Only cells below a certain size threshold can be sexualized, but various environmental factors can modulate the success of sexual reproduction. We investigated the role of cell density and physiological conditions of parental strains in affecting the success and timing of sexual reproduction in the marine heterothallic diatom Pseudo-nitzschia multistriata. We also studied the dynamics of the sexual phase in still conditions allowing cell sedimentation and in gently mixed conditions that keep cells in suspension. Our results showed that successful sexual reproduction can only be achieved when crossing parental strains in the exponential growth phase. Evidence was provided for the fact that sexual reproduction is a density-dependent event and requires a threshold cell concentration to start, although this might vary considerably amongst strains. Moreover, the onset of the sexual phase was coupled to a marked reduction in growth of the vegetative parental cells. The crosses carried out in physically mixed conditions produced a significantly reduced number of sexual stages as compared to crosses in still conditions, showing that mixing impairs sexualization. The results of our experiments suggest that the signaling that triggers the sexual phase is favored when cells can accumulate, reducing the distance between them and facilitating contacts and/or the perception of chemical cues. Information on the progression of the sexual phase in laboratory conditions help understanding the conditions at which sex occurs in the natural environment.

De novo transcriptome assembly of a lipoxygenase knock-down strain in the diatom Pseudo-nitzschia arenysensis.

Diatoms are microalgae that live in marine and freshwater environments and are responsible for about 20% of the world's carbon fixation. Population dynamics of these cells is finely regulated by intricate signal transduction systems, in which oxylipins are thought to play a relevant role. These are oxygenated fatty acids whose biosynthesis is initiated by a lipoxygenase enzyme (LOX) and are widely distributed in all phyla, including diatoms. Here, we present a de novo transcriptome obtained from the RNA-seq performed in the diatom species Pseudo-nitzschia arenysensis, using both a wild-type and a LOX-silenced strain, which will represent a reliable reference for comparative analyses within the Pseudo-nitzschia genus and at a broader taxonomic scale. Moreover, the RNA-seq data can be interrogated to go deeper into the oxylipins metabolic pathways.

Multiannual patterns of genetic structure and mating type ratios highlight the complex bloom dynamics of a marine planktonic diatom.

Understanding the genetic structure of populations and the processes responsible for its spatial and temporal dynamics is vital for assessing species' adaptability and survival in changing environments. We investigate the genetic fingerprinting of blooming populations of the marine diatom Pseudo-nitzschia multistriata in the Gulf of Naples (Mediterranean Sea) from 2008 to 2020. Strains were genotyped using microsatellite fingerprinting and natural samples were also analysed with Microsatellite Pool-seq Barcoding based on Illumina sequencing of microsatellite loci. Both approaches revealed a clonal expansion event in 2013 and a more stable genetic structure during 2017-2020 compared to previous years. The identification of a mating type (MT) determination gene allowed to assign MT to strains isolated over the years. MTs were generally at equilibrium with two notable exceptions, including the clonal bloom of 2013. The populations exhibited linkage equilibrium in most blooms, indicating that sexual reproduction leads to genetic homogenization. Our findings show that P. multistriata blooms exhibit a dynamic genetic and demographic composition over time, most probably determined by deeper-layer cell inocula. Occasional clonal expansions and MT imbalances can potentially affect the persistence and ecological success of planktonic diatoms.

The first genetic engineered system for ovothiol biosynthesis in diatoms reveals a mitochondrial localization for the sulfoxide synthase OvoA.

Diatoms represent one of the most abundant groups of microalgae in the ocean and are responsible for approximately 20% of photosynthetically fixed CO2 on Earth. Due to their complex evolutionary history and ability to adapt to different environments, diatoms are endowed with striking molecular biodiversity and unique metabolic activities. Their high growth rate and the possibility to optimize their biomass make them very promising 'biofactories' for biotechnological applications. Among bioactive compounds, diatoms can produce ovothiols, histidine-derivatives, endowed with unique antioxidant and anti-inflammatory properties, and occurring in many marine invertebrates, bacteria and pathogenic protozoa. However, the functional role of ovothiols biosynthesis in organisms remains almost unexplored. In this work, we have characterized the thiol fraction of Phaeodactylum tricornutum, providing the first evidence of the presence of ovothiol B in pennate diatoms. We have used P. tricornutum to overexpress the 5-histidylcysteine sulfoxide synthase ovoA, the gene encoding the key enzyme involved in ovothiol biosynthesis and we have discovered that OvoA localizes in the mitochondria, a finding that uncovers new concepts in cellular redox biochemistry. We have also obtained engineered biolistic clones that can produce higher amount of ovothiol B compared to wild-type cells, suggesting a new strategy for the eco-sustainable production of these molecules.

Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum.

The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.

Trade-off between sex and growth in diatoms: Molecular mechanisms and demographic implications.

Diatoms are fast-growing and winning competitors in aquatic environments, possibly due to optimized growth performance. However, their life cycles are complex, heteromorphic, and not fully understood. Here, we report on the fine control of cell growth and physiology during the sexual phase of the marine diatom Pseudo-nitzschia multistriata. We found that mating, under nutrient replete conditions, induces a prolonged growth arrest in parental cells. Transcriptomic analyses revealed down-regulation of genes related to major metabolic functions from the early phases of mating. Single-cell photophysiology also pinpointed an inhibition of photosynthesis and storage lipids accumulated in the arrested population, especially in gametes and zygotes. Numerical simulations revealed that growth arrest affects the balance between parental cells and their siblings, possibly favoring the new generation. Thus, in addition to resources availability, life cycle traits contribute to shaping the species ecological niches and must be considered to describe and understand the structure of plankton communities.

An optimised method for intact nuclei isolation from diatoms.

Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.

New alleles in the mating type determination region of West Atlantic strains of Pseudo-nitzschia multistriata.

The cosmopolitan, species-rich diatom genus Pseudo-nitzschia represents a good system for the study of speciation, evolution and diversity. Understanding elements linked to population dynamics and life cycle regulation for these species is of particular importance in view of their ability to produce the toxin domoic acid and cause harmful blooms. Pseudo-nitzschia multistriata, one of the toxic species that represents a model for the study of life cycle related questions, is the only diatom for which a sex determination mechanism has been described. Populations in the Gulf of Naples (Mediterranean Sea), can share four different allelic variants (A, M, B, N) of the mating type determination region, and one of them (A) is responsible for the determination of the mating type + (MT+), defined by the MT+ restricted expression of the gene MRP3. Here, we analysed the sex determination genomic region in three new strains isolated from the Gulf of Mexico and compared it to the alleles previously described in the Mediterranean strains. We first show that these geographically distant strains of P. multistriata belong to different populations but can interbreed. Next, we show that the two populations share an overall similar structure of the genomic locus although differences can be seen in the polymorphic regions upstream of MRP3. In strain P4-C1, we amplified and sequenced an allele (M) identical to one of those previously characterized in the Mediterranean strains. In the other two strains, P4-C2 and P4-C5, we identified three new alleles, which we named A2, B2 and N2. P4-C2 and P4-C5 are heterozygous and share the common allele A2 linked to the monoallelic expression of the MT+ specific sex determining gene MRP3. Our results expand information on the global distribution of P. multistriata and on the level of conservation of the sex determination region in different populations. The definition of the extent of intra- and inter-specific conservation of this region would be a relevant addition to our understanding of Pseudo-nitzschia diversity and evolution.